Atrial fibrillation (AF) is the most common arrhythmia affecting over 2 million Americans, and is associated with an increased risk of stroke, dementia, heart failure and death. Many common risk factors for AF have been identified, and in the past five years there have been increasing data supporting a genetic contribution to AF. Genetic loci and mutations for AF have been described predominately in ion channel proteins, though these variants are rare causes of AF. There remains a significant, but unexplained genetic basis for AF. A genome-wide association study (GWAS) in Icelanders has identified a susceptibility region for AF on Chr 4q25. Using 3,508 subjects with AF and 12,173 referent subjects from four additional centers, we have recently performed a replication and meta-analysis confirming these findings. Thus, the initial GWAS, replication, and meta-analysis have all demonstrated a convincing association between Chr 4q25 and AF. There are no known genes in the LD block containing these SNPs;however, the closest gene is a strong candidate gene for AF. PITX2 is a transcription factor that has a critical role in determining left-right asymmetry, and the development of the left atrium and pulmonary veins. Ectopic electrical foci within the pulmonary veins initiates fibrillatory activity and is the target of catheter ablation procedures used to treat AF. Recent studies have demonstrated that short regions of highly conserved intergenic DNA are often found adjacent to transcription factors and regulate gene function by acting as tissue specific enhancers. Given the lack of any genes in the LD block associated with AF, and an adjacent candidate gene necessary for left atrial and pulmonary vein development, we hypothesize that the SNPs associated with AF regulate PITX2 activity via highly conserved enhancers. In preliminary studies, we have performed an initial screen for enhancer elements in the LD block associated with AF. We have identified one such element, and found that SNPs in LD with this enhancer confer an independent risk for AF. We propose to extend this work through the following specific aims: Aim 1. To determine if the SNPs associated with AF regulate gene expression at the Chr 4q25 locus. Aim 2. To identify and characterize conserved non-coding enhancers at the Chr 4q25 locus for AF by: 2A. Using an in vivo zebrafish model system to rapidly identify conserved non-coding enhancers. 2B. Performing an in vitro screen for transcriptional regulators of PITX2 activity. 2C. Characterizing the identified transcriptional regulatory elements using a mammalian expression system. Identification of mechanism by which variation at this locus leads to AF will provide an opportunity to advance our understanding of the pathogenesis, risk stratification, therapeutic modalities for this common arrhythmia. PUBLIC HEALTH RELEVANCE: Atrial fibrillation is the most common abnormality of the heart rhythm and increases the risk of stroke and death. Genetic studies have identified a region of susceptibility for atrial fibrillation, but the mechanism by which genetic variation in this area leads to atrial fibrillation is unknown. We propose to screen this region of susceptibility in zebrafish, cell lines, and mice for functional elements that may lead to atrial fibrillation.